Modified human chorionic gonadotropin (β-hCG) proteins and their medical use

ABSTRACT

The present invention relates to modified human chorionic gonadotropin (β-hCG) proteins and their medical use as immunological contragestatives. The modification causes a reduction in the cross-reactivity of the modified β-hCG protein with luteinizing hormone (LH) as defined by the ability of both proteins to react with the same antibody.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 filing of International ApplicationNo. PCT/GB96/01717, filed Jul. 19, 1996.

FIELD OF THE INVENTION

The present invention relates to substances, in particular to modifiedhuman chorionic gonadotropin (β-hCG) proteins/genes, and their medicaluse, for example as immunological contraceptives having improvedspecificity and/or which in vivo avoid producing antibodies havingundesirable cross-reactivity, for example with other natural hormones.

BACKGROUND OF THE INVENTION

The principle of immunising the female with β-hCG or its C-terminalpeptide to induce antibodies which neutralise hCG and therefore inhibitpregnancy has been proposed¹ and has been the subject of trials by theWorld Health Organization² and the Indian Health Authorities³.

Shortly after fertilization of the ovum, the hormone hCG which at othertimes is essentially absent from the body, is produced and acts on thecorpus luteum in the ovary to promote synthesis of progesterone.Progesterone is vital for the maintenance of the fertilized egg in theuterus and so the production of antibodies to neutralise the hCG willeffectively prevent the pregnancy from proceeding. This strategy hasbeen successfully employed to block fertility in baboons¹ and marmosets⁴and more recently in humans³.

hCG itself is composed of two chains, α and β. The α-chain is common toother hormones (FSH, TSH and LH), which contribute to normalphysiological function, so that autoantibodies made to this chain wouldbe highly undesirable. The β-chain of hCG is far more specific, but amajor problem still remains in that there is an 85% homology of β-hCGwith the β-chain of luteinizing hormone (LH) which is presentcontinually in the potentially fertile female. A strategy adopted by theW.H.O. has been to prepare a vaccine based on the β-hCG C-terminalpeptide (residues 109-145) which is unique to hCG. This is madeimmunogenic by linking to the carrier proteins tetanus or diphtheriatoxoids to provide T-cell help. This has not produced adequately highresponses in high frequency within the cohorts tested⁵ partly because ofthe relatively weak immunogenicity of the peptide and the fact thatantibodies to a peptide fragment of a protein do not usually bind withhigh affinity to the parent protein⁶.

Talwar adopted a less cautious approach by using the whole β-hCG chain(together with ovine α-chain as a carrier) in the hope that theantibodies produced which cross-reacted with LH would not prove to betroublesome. However, not enough experience has been gained so far toconfirm this hope and in principle, where possibly millions of peoplecould be immunized with the vaccine for several years, it would seemprudent, to devise a vaccine which did not cross-react with LH.

SUMMARY OF THE INVENTION

It is known that the epitopes specific for β-hCG other than theC-terminus are discontinuous, i.e. the residues making up the epitopemay be separate from each other in primary structure but are broughttogether by the protein folding. However, the contact residues formingthese discontinuous epitopes are very difficult to identify and even ifthey could be, the “floppiness” of any synthetic peptide formed fromthese residues would make it a poor immunogen with respect to thegeneration of antibodies with high affinity.

In the present invention, we have adopted a strategy⁷ which relies uponthe natural folding of the protein to form the specific discontinuousepitope, while at the same time mutating the parent gene in such a waythat the amino acid residues forming the LH cross-reacting epitopes arealtered without affecting the more distant folding of the hCG-specificepitope(s). The retention of the desired epitope(s) and the loss of theunwanted epitopes can be monitored by reaction of the mutants withmonoclonal antibodies specific for hCG and others giving cross-reactionbetween hCG and LH.

Broadly, the present invention provides a substance which has theproperty of inducing a neutralising antibody response to β-hCG in vivo,said antibodies not substantially cross-reacting with LH, the substancecomprising one or more of the conformational epitopes specific to nativeβ-hCG, or functional equivalents or mimetics of these epitopes.

In one aspect, the substance is a modified β-hCG protein having one ormore conformational epitopes specific to native β-hCG, the protein beingmodified at one or more amino acid residues forming epitope(s) of nativeβ-hCG that cross-react with LH, to reduce the cross-reactivity the β-hCGprotein with LH, as defined by the ability of both proteins to reactwith the same antibody. The present invention also includes substanceswhich are variants, derivatives, functional equivalents or mimetics ofthese above proteins.

Preferably, the substance includes two or more epitopes that arespecific to native β-hCG. This helps to induce the production ofantibodies specific for these epitopes, which will form complexes of theβ-hCG with two or more antibody molecules, so helping to improve the invivo neutralising activity caused by the substance.

Preferably, the modified amino acid residues are selected from thefollowing residues of native β-hCG; Lys20, Glu21, Gly22, Pro24, Val25,Glu65, Arg68, Gly71, Arg74, Gly75 and/or Val79.

There are other residues common to β-hCG and β-LH which lie on theoutside of the protein molecule accessible to the aqueous solvent phase,which might potentially be immunogenic and give rise to cross-reactingantibodies. This would have to be established following immunisationwith the mutant β-hCG and a similar further mutation procedure wouldthen be required to abolish the epitopes reacting with these newantibodies.

The rationale for selecting the residues to replace the native residuesis set out below in more detail. Preferred modifications are set out intable 2.

In a further aspect, the present invention provides nucleic acidencoding the above proteins, vectors incorporating the nucleic acid andhost cells transformed with the vectors.

In a further aspect, the present invention includes compositionscomprising one or more of the above substances, in combination with aphysiologically acceptable carrier. Preferably, the compositions will becontraceptive compositions in a form suitable for immunisation. However,the substances, proteins or compositions described herein may proveuseful in hCG-specific immunoassays and for applications where hCG isactive, such as the inhibition of Kaposi sarcoma.

In a further aspect, the present invention provides a method ofcontraception, more strictly in this context contragestative, for afemale mammal comprising immunising the female mammal with acontraceptively effective amount of one or more of the substances.

In a further aspect, the present invention includes the use of thesubstances in the manufacture of a contraceptive composition.

Conveniently, the immunogenicity of the substance may be enhanced bylinking it to a carrier such as tetanus toxoid, or to appropriatesequences from such a carrier acting as T-helper epitopes. Additionallythe substance may be engineered as a fusion protein with anappropriately immunogenic partner. Engineered DNA constructs containingnucleotide sequences encoding the substance together with, for example,additional sequences encoding T-helper epitopes or cytokine adjuvants,may be directly administered as a nucleic acid, preferably DNA, vaccine.

It will be appreciated that the nucleic acid construct encoding themodified β-hCG protein can be used as an initial vaccine to prime animmune response. This initial response can then be boosted by subsequentinjection of the modified β-hCG protein itself. Likewise, the modifiedβ-hCG protein could be used first followed by the nucleic acid to boostthe immune response.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesise or where it is unsuitable for a particularmethod of administration, eg peptides are unsuitable active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing is generallyused to avoid randomly screening large number of molecules for a targetproperty.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,eg by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, eg stereochemistry, bonding, sizeand/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

The present invention will now be described in more detail by way ofexample with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically a modified β-hCG protein in which an epitopewhich cross-reacts with LH is modified; more particularly an epitopedeletion mutant which has lost epitope (▪) cross-reacting with β-LH butstill retains β-hCG specific epitope ().

FIG. 2 comprises FIGS. 2A-2D, and shows the fluorescence of celltransfected with β-hCG mutant 6 determined by Facscan: 2A-controlantibody; 2B-stained by anti-C terminal epitope; 2C-stained byanti-hCG-specific β₁ epitope; 2D-stained with antibody to LHcross-reacting β₃ epitope showing loss of staining;

FIG. 3 shows the relative spatial distribution of the β-hCG epitopeclusters;

FIG. 4 shows the results of the binding of Mabs to different mutants;and,

FIG. 5 shows the amino acid substitutions made in β-hCG.

DETAILED DESCRIPTION

Production of Mutant β-hCG

β-hCG has a size of 145 amino acid residues which include 12 cysteinesthat form 6 conserved di-sulphide bridges. The subunit is heavilyglycosylated with N-linked carbohydrates at position Asn13 and Asn30inaddition to four O-linked carbohydrates in the C-terminus at Ser121,127, 132 and 138. To ensure correct folding of the recombinant moleculeswe opted for expression in mammalian cells. Using a construct whereβ-hCG is synthesized as a fusion protein with a C-terminal extensionthat consists of the 17 amino acids proximal to the membrane, thetransmembrane and the cytoplasmic portion of the H2-D^(b) molecule, itwas possible to express wild type and mutant hCG on the surface oftransfected cells. The expression level was determined with the hCGspecific Mab OT3A on a Becton Dickinson Facscan as shown in FIG. 2. TheMab OT3A which recognizes a linear epitope in the C-terminal extensionof β-hCG can be used to quantitate the expression level of the wild typeand mutant recombinant proteins following transient transfection in theCOS7 cells.

Berger et al^(8&9) have previously used a panel of Mab to define 8separate epitope clusters on β-hCG, which can graphically be related toeach other using a cylindrical Mercator's projection (FIG. 3). Theβ1-β5, β8 and β9 clusters are present on the heterodimeric hCGholohormone, whereas β6 and β7 are unique to the free β-hCG subunit. Theβ1, β6, β7, β8 and β9 are unique to hCG, whereas the Mab to the otherepitope clusters cross-react with βLH. All the Mabs used apart fromOT3A, recognize discontinuous sequences on β-hCG, because reduction andalkylation of β-hCG abolishes the binding of the Mab⁹. As summarised inTable 1 (FIG. 4) all the Mab against the different β-hCG epitopeclusters used in this study bind to surface expressed wild type β-hCG,transiently expressed in COS7 cells. This suggests that the folding ofthe recombinant wild type β-hCG is as seen in the native molecule.

The target residues for mutation were selected from the crystalstructure of hCG to have side chains protruding from the surface of themolecule which could contribute to the antibody binding site. Toincrease the likelihood for correct folding of the mutants thesubstitutions were selected by comparing the same residues in thedifferent members of the same family. The changes were designed,however, to introduce amino acids with sufficiently dissimilarproperties in their side chains (e.g. charge, size, polarity) from theβ-hCG residues, to disrupt any Mab binding in this region. Computergraphic model building of the mutant β-hCG molecules ensured that theside chains of the amino acid substitutions could be accommodated intothe predicted structure without grossly altering the overallconformation. Table 2 (FIG. 5) summarizes the amino acid changes ineleven of the mutants used in this study.

Expression Vector Construct and Production of Mutants

Full length β-hCG cDNA was cloned from human placental third trimesterRNA using RT-PCR and the sense cloning primer5′ACCGGAATTCCAGGGGCTCCTGCTGTTG3′ (SEQ ID NO:1)(corresponding tonucleotide(nt) −51→−33) and the antisense cloning primer5′TTGGTCGACTTGTGGGAGGATCGGGGTGTCC3′ (SEQ ID NO:2)(nt 414→435). The hCGcDNA was cloned into pCDM8¹⁰ into which a DNA fragment from H2-Dbcontaining the 17 membrane proximal amino acid residues, thetransmembrane region and cytoplasmic tail had been inserted. Thisfragment was obtained using RT-PCR amplification using RNA from a spleenof a C57BL/10 mouse with the sense primer5′GCGTTGGTCGACCATGAGGGGCTGCCTGAGCCC3′ (SEQ ID NO:3)(nt 547→566) and anantisense primer 5′CACAGGAGAGACCTGAACACATCG3′ (SEQ ID NO:4)(nt 809→832).The sequence of β-hCG is as published¹¹.

The mutants were produced by an overlap PCR mutagenesis method¹².Examples of primer sequences that were used include:

mutant 1

sense 5′GAGAACCGCGAGTGCCCCGTGTGCATCACCGTC3′ (SEQ ID NO:5);

antisense 5′GGCACTCGCGGTTCTCCACAGCCAGGGTGGC3′ (SEQ ID NO:6);

mutant 2

sense 5′CCACTACTGCATCACCGTCAACACCACCATGTGCC3′ (SEQ ID NO:7);

antisense 5′CGGTGATGCAGTAGTGGCAGCCCTCCTTCTCC3′ (SEQ ID NO:8);

mutant 3

sense 5′GGCTGCCCCTCCCACGTGAACCCCCACGTCTCCTACGCCGTG3′ (SEQ ID NO:9);

antisense 5′CGTGGGAGGGGCAGCCAGGGAGCTCGATGGACTCGAAG3′ (SEQ ID NO:10);

mutant 4

sense 5′GGAGAACCGCGAGTGCCACTACTGCATCACCGTCAAC3′ (SEQ ID NO:11);

antisense 5′GACGGTGATGCACACGTGGCAGCCCTCCTTCTC3′ (SEQ ID NO:12);

mutant 5

sense 5′GAGAAGGAGGGCTGCCACGTGTGCATCACCGTC3′ (SEQ ID NO:13);

antisense 5′GACGGTGATGCACACGTGGCAGCCCTCCTTCTC3′ (SEQ ID NO:14);

mutant 6

sense 5′GAAGGAGGGCTGCCCCTACTGCATCACCGTCAAC3′ (SEQ ID NO:15);

antisense 5′GTTGACGGTGATGCAGTAGGGGCAGCCCTCCTTC3′ (SEQ ID NO:16).

β-hCG, or the mutants themselves, were used to generate mutants/furthermutants.

The sequence of all the mutations were verified using double strandedDNA sequencing (Sequenase USB) and a range of β-hCG internal and CDM8primers.

Transfections, Surface Expression, Staining and FACs Analysis

COS cells were transfected using a modified DEAE dextran-chloroquinemethod (based on Seed & Aruffo¹³). Briefly, 1.5×10⁶ cells were seededinto an 80 cm³ flask on the day before transfection. 6 ml of thetransfection mixture, (10% NuSerum (Becton Dickinson, Bedford Mass.);1-2μg/ml supercoiled DNA (CsCl prepared or PEG prepared); 250 μg/ml DEAEdextran) was added to the washed monolayer and left in 37° C. incubatorfor 60 minutes. Chloroquine was then added to a final concentration of200 μM and the cells incubated for a further 120 minutes. Thetransfection mixture was then removed, the monolayer washed with PBS and3 ml 10%DMSO (in PBS) added for 2 minutes. The cells were washed againand complete medium added. The cells were split 1:1 24 hours later andharvested 65-72 hours after transfection. A transfection efficiency of20-40% was routinely obtained.

Cells were stained prior to Facs analysis in duplicates of 2×10⁵ cellsfor each Mab tested. Following washing of the harvested cells with PBS;10% FCS; 0.02% NaN₃. They were incubated with 100 μl of theconformation-dependent anti-β-hCG Mab for 30 minutes on ice, washedtwice in PBS 0.02% NaN₃ and then incubated with 100 μl of rabbitanti-mouse Fc Flourescein isothyocyanate conjugate. Following washingthe cells were fixed in 1% formaldehyde in PBS, and Facs analysisperformed using a Becton-Dickinson Facscan. Markers were set on thenegative control which was routinely an anti-CD34 IgG1. All cells to theright of this marker were deemed to be positively transfected.

Results

The results of staining wild type and mutant β-hCG expressed on thesurface of COS7 cells with the panel of Mabs are summarized in Table 1 .The Mabs to the β1 epitope cluster and the Mab OT3A bind to wild typeand all the mutant β-hCG with the same relative binding. Thisdemonstrates that the mutant molecules fold to completely recreate thehCG specific epitope β1. The mutations in the N-terminal hairpin loop(Lys20, Glu21, Gly22, Pro24 and Val25) completely abolish binding of theMab specific for the β3 and β6 epitope cluster, and lead to partialbinding of Mab 3E2 specific for the β3/5 cluster. Different mutants weremade to pinpoint the important amino acids that contribute to thebinding of the different Mabs.

Mutations of residues Lys20-Glu2-Gly22 (Mutant 1) completely abolish thebinding of the Mab InnhCG64 recognizing the hCG-specific epitope clusterβ6 and lead to partial binding of the β3/5 Mab 3E2. Mutant 2(Pro24-Val25) fails to bind both β3 specific Mabs (InnLH1 and InnhCG111)and also reduces binding of 3E2 to 25-50%. The two β3 Mabs have separatebut overlapping binding sites on β-hCG, because a single point mutationPro24→His (Mutant 5) completely abolishes binding of Mab InnLH1 butallows partial binding of InnhCG111 (63%), whereas the mutationVal25→Tyr (Mutant 6) prevents binding of InnhCG111 and reduces thebinding of InnLH1 to 63%. Combining all five point mutations of theN-terminal hairpin loop (Mutant 4) is required to reduce the binding of3E2 to 13% compared to that of OT3A.

In contrast to this the four mutations at residues 68, 74, 75, 79introduced in the C-terminal hairpin loop (Mutant 3) completely abolishthe binding of all cross-reactive antibodies to the mutated molecule yetretain the binding of the hCG-specific Mabs directed to the β1 and β7epitope clusters and to the linear epitope in the C-terminus of MabOT3A.

Discussion

The strategy of producing epitope-specific vaccines by allowing thenatural folding of a protein to retain a desired discontinuous epitopewhile at the same time removing unwanted epitopes by mutation, isclearly feasible. It has proved possible to construct mutants whichstill display epitopes specific for β-hCG, even though they have lostepitopes cross-reacting with luteinizing hormone with which the parentmolecule shares 85% homology. Retention of the ability of the mutants toreact with the β-hCG-specific monoclonals implies that the structuralchanges introduced into the molecule have not affected the tertiaryfolding of the chains which generate the related epitope since this isknown to have a discontinuous structure.

The design of the mutants was guided by three main principles. Residuesselected for mutation should contribute to epitope formation, theyshould be common to β-hCG and the cross-reacting luteinizing hormone,and their modification should not significantly influence the overallfolding of the molecule at distant sites. The successful deletionmutants clearly achieved the primary objective of preserving thefunctional structure of the β₁-specific epitope but considerableprogress has also been made towards the secondary aim of identifying thelocation of the epitopes themselves.

We have shown that, even with a molecule like hCG which has complexnon-contiguous B-cell epitopes, it is possible to make radical changesin structure which remove unwanted epitopes yet maintain other desirableepitopes. It seems likely that the many different amino acidsubstitutions will provide good T-cell helper epitopes. Alternatively,the mutant can either be linked to a carrier such as tetanus toxoid, orengineered as a fusion protein with an appropriately immunogenicpartner.

REFERENCES

1. Stevens, V. C., Powell, J. E., Lee, A. C. and Griffin, P. D.Anti-fertility effects from immunization of female baboons withC-terminal peptides of human chorionic gonadotrophin. Fertil Steril.,1981, 36, 98-105.

2. Jones W. R., Judd, S. J., Ing, R. M. Y., Powell, J., Bradley, J.,Denholm, E. H., Mueller, U. W., Griffin, P. D., and Stevens, V. C. PhaseI clinical trials of a world health organisation birth control vaccine.Lancet, Jun. 4, 1988, 1295-1298.

3. Talwar-GP; Singh-O; Pal-R; Chatterjee-N; Sahai-P; Dhall-K; Kaur-J;Das-SK; Suri-S; Buckshee-K; et-al. A vaccine that prevents pregnancy inwomen. Proc-Natl-Acad-Sci-U-S-A. Aug. 30, 1994, 91(18), 8532-6.

4. Hearn, J. B. Immunisation against pregnancy. Proc R Soc B., 1976;195, 149-60.

5. Dirnhofer, S., Klieber, R., De Leeuw, R., Bidart, J. M., Merz, W. E.,Wick, G. and Berger, P. Functional and immunological relevance of theCOOH-terminal extension of human chorionic gonadortropin β: implicationsfor the WHO birth control vaccine. FASEB, 1993, 7, 1382-1385.

6. Roitt I. M. Essential Immunology, 8th Edition. Blackwell ScientificPublications, 1994, p. 281.

7. Roitt, I. M. Basic concepts and new aspects of vaccine developmentParasitology, 1989, 98 S7-S12.

8. Berger, P., Klieber, R, Panmoung, W., Madersbacher, S., Wolf. H. andWick, G. Monoclonal antibodes against the free subunits of humanchorionic gonadotrophin. J. Endocrinology, 1990, 125, 301-309.

9. Dirnhofer, S., Madersbacher, S., Bidart J-M., Ten, P. B. W.,Kortenaar Spottie G., Mann, K., Wick, G. and Berger, P. The molecularbasis for epitopes on the free β subunit of human chorionicgonadotrophin, its carboxyl terminal peptide and the hCG core fragmentJ. of Endocrinology, 1994, 121,153-162.

10. Seed, B. An LFA-3 cDNA encodes a phospholipid-linked membraneprotein homologous to its receptor CD2. Nature (Lond.), 1987, 329,840-842.

11. Talmadge, K, Vamvakopoulos, N. C. and Fiddes, J. C. Evolution of thegenes for the beta subunits of human chorionic gonadotropin andluteinizing hormone. Nature. 1984, 307, 37-40.

12. Horton, R. M. and Pease, L. R. Recombination and mutagenesis of DNAsequences using PCR. from Directed Mutagenesis: A Practical Approach, EdMcPherson, M. J. IRL Press, 1991, 217-247.

13. Seed, B., and Aruffo, A. Molecular cloning of the human CD2 antigenby a rapid immunoselection procedure. Proc Natl Acad Sci. USA, 1987, 84,3365-3369.

16 1 28 DNA Artificial Sequence Description of Artificial SequenceBeta-hcG Human Sense Cloning Primer 1 accggaattc caggggctcc tgctgttg 282 31 DNA Artificial Sequence Description of Artificial Sequence Beta-hcGHuman Antisense Cloning Primer 2 ttggtcgact tgtgggagga tcggggtgtc c 31 333 DNA Artificial Sequence Description of Artificial Sequence C57BL/10Mouse Sense Primer 3 gcgttggtcg accatgaggg gctgcctgag ccc 33 4 24 DNAArtificial Sequence Description of Artificial Sequence C57BL/10 MouseAntisense Primer 4 cacaggagag acctgaacac atcg 24 5 33 DNA ArtificialSequence Description of Artificial Sequence Mutant 1 Sense Primer 5gagaaccgcg agtgccccgt gtgcatcacc gtc 33 6 31 DNA Artificial SequenceDescription of Artificial Sequence Mutant 1 Antisense Primer 6ggcactcgcg gttctccaca gccagggtgg c 31 7 35 DNA Artificial SequenceDescription of Artificial Sequence Mutant 2 Sense Primer 7 ccactactgcatcaccgtca acaccaccat gtgcc 35 8 32 DNA Artificial Sequence Descriptionof Artificial Sequence Mutant 2 Antisense Primer 8 cggtgatgca gtagtggcagccctccttct cc 32 9 42 DNA Artificial Sequence Description of ArtificialSequence Mutant 3 Sense Primer 9 ggctgcccct cccacgtgaa cccccacgtctcctacgccg tg 42 10 38 DNA Artificial Sequence Description of ArtificialSequence Mutant 3 Antisense Primer 10 cgtgggaggg gcagccaggg agctcgatggactcgaag 38 11 37 DNA Artificial Sequence Description of ArtificialSequence Mutant 4 Sense Primer 11 ggagaaccgc gagtgccact actgcatcaccgtcaac 37 12 33 DNA Artificial Sequence Description of ArtificialSequence Mutant 4 Antisense Primer 12 gacggtgatg cacacgtggc agccctccttctc 33 13 33 DNA Artificial Sequence Description of Artificial SequenceMutant 5 Sense Primer 13 gagaaggagg gctgccacgt gtgcatcacc gtc 33 14 33DNA Artificial Sequence Description of Artificial Sequence Mutant 5Antisense Primer 14 gacggtgatg cacacgtggc agccctcctt ctc 33 15 34 DNAArtificial Sequence Description of Artificial Sequence Mutant 6 SensePrimer 15 gaaggagggc tgcccctact gcatcaccgt caac 34 16 34 DNA ArtificialSequence Description of Artificial Sequence Mutant 6 Antisense Primer 16gttgacggtg atgcagtagg ggcagccctc cttc 34

What is claimed is:
 1. A modified β-hCG protein, the protein amino acidsequence being modified by recombinant means by one or more amino acidsubstitutions selected from the group consisting of 20 (Lys) to Asn, 21(Glu) to Arg and 22 (Gly) to Glu; 24 (Pro) to His; 25 (Val) to Tyr; 68(Arg) to Glu; 74 (Arg) to Ser; 75 (Gly) to His; 79 (Val) to His; and 71(Gly) to Arg and 74 (Arg) to Ser; so as to reduce the cross-reactivityof the modified β-hCG protein with LH as defined by the ability of bothproteins to react with the same antibody, wherein the modified β-hCGprotein retains one or more conformational epitopes specific to thenative β-hCG.
 2. The modified β-hCG protein according to claim 1 beingmodified by at least amino acid substitution 68 (Arg) to Glu.
 3. Themodified β-hCG protein according to claim 1 being modified by at leastamino acid substitution 74 (Arg) to Ser.
 4. The modified β-hCG proteinaccording to claim 1 being modified by at least amino acid substitution20 (Lys) to Asn, 21 (Glu) to Arg and 22 (Gly) to Glu.
 5. The modifiedβ-hCG protein according to claim 1 being modified by at least amino acidsubstitution 24 (Pro) to His.
 6. The modified β-hCG protein according toclaim 1 being modified by at least amino acid substitution 25 (Val) toTyr.
 7. The modified β-hCG protein according to claim 1 being modifiedby at least amino acid substitution 71 (Gly) to Arg, and 74 (Arg) toSer.
 8. The modified β-hCG protein according to claim 1 being modifiedby at least amino acid substitution 75 (Gly) to His.
 9. The modifiedβ-hCG protein according to claim 1 being modified by at least amino acidsubstitution 79 (Val) to His.
 10. The modified β-hCG protein accordingto claim 1 wherein the protein amino acid sequence is modified by pointmutation.
 11. The modified β-hCG protein according to claim 1 whereinthe modified β-hCG protein is chemically linked by a chemical linkage toan immunogenic substance.
 12. The modified β-HCG protein according toclaim 11 wherein the chemical linkage is by co-expression as a fusionprotein.
 13. An isolated and purified nucleic acid sequence encoding amodified β-hCG protein according to claim
 1. 14. The isolated andpurified nucleic acid according to claim 13 further encoding a fusionprotein comprising an immunogenic carrier protein fused to the modifiedβ-hCG protein.
 15. An expression vector comprising a nucleic acidaccording to claim
 13. 16. An expression vector comprising a nucleicacid according to claim
 14. 17. A mammalian host cell comprising avector according to claim
 15. 18. A mammalian host cell comprising avector according to claim
 16. 19. A mammalian host cell comprisingnucleic acid according to claim
 13. 20. A mammalian host cell comprisingnucleic acid according to claim
 14. 21. A microbial host cell comprisinga vector according to claim
 15. 22. A microbial host cell comprising avector according to claim
 16. 23. A microbial host cell comprisingnucleic acid according to claim
 13. 24. A microbial host cell comprisingnucleic acid according to claim
 14. 25. A composition comprising amodified β-hCG protein according to claim 1 and a pharmaceuticallyacceptable carrier.
 26. A composition comprising a nucleic acidaccording to claim 13 and a pharmaceutically acceptable carrier.
 27. Acomposition comprising a nucleic acid according to claim 14 and apharmaceutically acceptable carrier.
 28. A composition comprising anexpression vector according to claim 15 and a pharmaceuticallyacceptable carrier.
 29. A composition comprising an expression vectoraccording to claim 16 and a pharmaceutically acceptable carrier.
 30. Acontragestative composition, comprising a modified β-hCG proteinaccording to claim 1 and a pharmaceutically acceptable carrier.
 31. Amodified β-hCG protein having a contragestative function in a femalemammal, wherein the modified β-hCG protein has an amino acid sequencethat is modified by recombinant means by one or more amino acidsubstitutions selected from the group consisting of 20 (Lys) to Asn, 21(Glu) to Arg and 22 (Gly) to Glu; 24 (Pro) to His; 25 (Val) to Tyr; 68(Arg) to Glu; 74 (Arg) to Ser; 75 (Gly) to His; 79 (Val) to His; and 71(Gly) to Arg and 74 (Arg) to Ser; so as to reduce the cross-reactivityof the modified β-hCG protein with LH as defined by the ability of bothproteins to react with the same antibody, wherein the modified β-hCGprotein retains one or more conformational epitopes specific to thenative β-hCG.
 32. A modified β-hCG protein according to claim 31 beingmodified by at least an amino acid substitution 68 (Arg) to Glu.
 33. Amodified β-hCG protein according to claim 31 being modified by at leastan amino acid substitution 74 (Arg) to Ser.